The present invention relates to a method for evaluating a compound exhibiting matrix metalloproteinase-inhibitory activity for an in vivo matrix metalloproteinase-inhibitory activity displayed by the compound, and specifically to a method therefor which comprises utilizing in situ zymography.
Matrix metalloproteinase (MMP) is a common name of enzymes which are secreted by cancer cells or normal cells surrounding cancers, and degrade extracellular matrix proteins such as collagen in a divalent metal ions-dependent manner. In particular, it has been frequently reported that type IV collagenases (MMP-2, MMP-9) are responsible for vascularization accompanied by tumor proliferation, and for tumor invasion and metastasis.
As an assay system for measuring matrix metalloproteinase, a gelatin zymography method is known wherein enzyme activities are determined based on degradation degree of a substrate. The method requires conducting electrophoresis, and therefore it is troublesome in the procedures, and time-consuming in the detection.
As an assay system without electrophoresis, The FASEB Journal, vol.9, July, pp.974-980, 1995 has proposed a determination method based on the principle of zymography, which comprises incorporating, into agarose, casein or gelatin bound to a fluorescent compound useful as a substrate for the protease, forming the mixture into a thin membrane on slide glass, then placing a tissue section on the surface of a thin membrane, incubating the membrane, and observing the digestion of the substrate by a fluorescence microscopy. The system, however, encounters a trouble with decreased reproducibility, since the agarose affects the digestion of the substrate due to the protease.
Recently, the applicants of the present application proposed a convenient and accurate method for measuring proteases, which comprises contacting a sample containing a protease to a thin membrane formed on the surface of a support, said thin membrane comprising a protease substrate and a hardening agent (WO97/32035), which have received some signs of confidence. For example, Proceedings of 5th Conference of the Japanese Association for Metastasis Research (1996) describes that in situ zymography based on our method was used to detect the gelatinase activity in the tissue and cells, thus observing that the protease destroyed tumor tissues. In situ zymography is described in The FEBS Journal, 974-980, Vol. 9 (1995) or the like.
Further, H. Nakamura, et al. Cancer Research 59,467-473(1999) describes that in situ zymography similarly revealed that MMP inhibitors inhibited the MMP activity in human thyroid cancer.
As described above, MMPs, in particular type IV collagenase, are believed to be responsible for vascularization accompanied by tumor proliferation, and for tumor invasion and metastasis. Accordingly, inhibitors of MMPs have been a focus of attention in view of development of anti-cancer agents, and, in fact, some of them are clinically being tested by some pharmaceutical companies.
Pharmacotherapy is achieved on the prerequisite that a drug used in the therapy is effective and safe, and the prerequisite is confirmed by drug evaluation, which is largely classified into two tests, nonclinical test such as animal experimentation, and clinical test wherein human beings are tested. Even agents that are effective in the nonclinical test, or have an excellent action mechanism would not be drugs when the agents are not effective in patients. This is the reason why the clinical test is required to evaluate the drug efficacy in humans.
In drug evaluation of MMP inhibitors by the clinical test, it is difficult to determine exactly the in vivo activity of MMP inhibitors because MMP is inherently controlled in vivo by endogenous inhibitors such as TIMP-1, and -2 [Woessner, J. F. Jr., FASEB J. 5, 2145-54 (1991)]. Immunostaining method wherein an antibody specific to MMP is used has been known as a method for detecting MMP (Int. J. Cancer., 56, 500-505 (1994)). Since the antibody used therein, however, recognizes MMP in a manner that MMP is merely an antigen protein, this method dose not allow to determine whether an antigen protein recognized by the antibody possesses the MMP activity or is inactivated by the inhibitors. Conventional gelatin zymography dissociates MMP from inhibitors during the electrophoresis, and therefore the method does not also allow the exact determination of the inhibitory activity of MMP inhibitors.
In view of the above, the present inventors investigated for a method of checking the in vivo activity of MMP inhibitors, and found that in situ zmography makes it possible to determine conveniently and rapidly the in vivo MMP inhibitory activity of MMP inhibitors.
In the first aspect, the present invention relates to a method for evaluating a compound exhibiting matrix metalloproteinase-inhibitory activity for an in vivo matrix metalloproteinase-inhibitory activity displayed by the compound, which comprises subjecting to in situ zymography a specimen isolated from a mammal that has received the compound.
Specifically, the present invention relates to the method wherein the in situ zymography is conducted using a thin membrane which comprises gelatin or collagen together with a hardening agent, and which has a thickness of 1 xcexcm to 20 xcexcm. More specifically, the in situ zymography is conducted by
(1) contacting the isolated specimen onto the thin membrane comprising a substrate of matrix metalloproteinase;
(2) incubating the specimen-thin membrane sample;
(3) staining and then decolorizing the thin membrane; and
(4) verifying the tone developed on the thin membrane.
In the evaluation method of the present invention, it is preferred that the substrate of matrix metalloproteinase is gelatin or collagen; that the thin membrane comprises a hardening agent; that the thin membrane has a thickness of 1 xcexcm to 20 xcexcm; and/or that the specimen is a tissue section, preferably a lyophilized tissue section, having a thickness of 2 xcexcm to 10 xcexcm, and the incubation is conducted at 20xc2x0 C. to 50xc2x0 C., preferably 36 to 38xc2x0 C., for 2 to 18 hours.
In the second aspect, the present invention relates to a method for evaluating a compound exhibiting matrix metalloproteinase-inhibitory activity for an in vivo matrix metalloproteinase-inhibitory activity displayed by the compound, which comprises:
(1) administering to a mammal a compound exhibiting matrix metalloproteinase-inhibitory activity; (2) isolating a tissue or a fluid from the mammal to prepare a specimen; (3) contacting the isolated specimen onto a thin membrane comprising a substrate of matrix metalloproteinase; (4) incubating the specimen-thin membrane sample; (5) staining and then decolorizing the thin membrane; and (6) verifying the tone developed on the thin membrane.
In the third aspect, the present invention relates to a method for evaluating an anti-cancer agent exhibiting matrix metalloproteinase-inhibitory activity for its anti-cancer activity, which comprises subjecting to in situ zymography a specimen isolated from a mammal that has received the anti-cancer agent, and evaluating for an in vivo matrix metalloproteinase-inhibitory activity displayed by the agent as an indicator of the anti-cancer activity. In the method of the present invention, it is preferred that the substrate of matrix metalloproteinase is gelatin or collagen; that the thin membrane comprises a hardening agent; that the thin membrane has a thickness of 1 xcexcm to 20 xcexcm; and/or that the specimen is a tissue section, preferably a lyophilized tissue section, having a thickness of 2 xcexcm to 10 xcexcm, and the incubation is conducted at 20xc2x0 C. to 50xc2x0 C., preferably 36 to 38xc2x0 C., for 2 to 18 hours.
It has been known that not only MMP but also many enzymes that degrade gelatin, such as trypsin, exist in the living body. For this reason, it was difficult to determine selectively the activity of MMP inhibitors by manes of in situ zymography using substrates of MMP such as gelatin, as used in the present invention. No method for determining an in vivo matrix metalloproteinase-inhibitory activity of MMP inhibitors has been known.
As used herein, xe2x80x9cin situ zymographyxe2x80x9d means an in vitro method useful for determination of protease activities, which is not involved in electrophoresis. In the present invention, this is conducted by contacting onto a thin membrane comprising a substrate of matrix metalloproteinase an specimen isolated from a mammal receiving a compound exhibiting matrix metalloproteinase-inhibitory activity (MMP inhibitor), and observing the MMP substrate after the lapse of a defined period of time, so that the inhibitor is evaluated for xe2x80x9cin vivo MMP inhibitory activityxe2x80x9d.
The term xe2x80x9cmammalxe2x80x9d in the phrase xe2x80x9cspecimen isolated from a mammalxe2x80x9d means a higher vertebrate, preferably human being, which has been administered with a MMP inhibitor. The test object in this method is a specimen isolated from the living body, which provides a result exactly corresponding to an in vivo MMP inhibitory activity of a MMP inhibitor, which the inhibitor exerts in vivo.
xe2x80x9cSpecimen isolatedxe2x80x9d may be derived from various tissues and fluids, and is varied depending on efficacy or indication of MMP inhibitors to be administered into a mammal. When, for example, a MMP inhibitor is for an anti-cancer agent, a specimen is derived from cancerous tissue samples isolated and taken by surgical operation or histological examination from cancerous tissues such as breast cancer, endometrioma, cancer of buccal cavity, lung cancer, gastric cancer, esophageal cancer, epithelial cancer of urinary tract, colon cancer, squamous cancer, thyroid cancer. For an anti-rheumatic agent, synovial membranes or fluids, or bone tissues derived from patients with rheumatoid arthritis or arthritis are used, whereas for an agent for treating heart failure, heart tissues or effusions therefrom derived from patients with heart failure would be used. In this manner, the present invention may be utilized as methods for evaluating anti-cancer agents, anti-rheumatic agents, and agents for treating heart failure.